ABSTRACT Gram-negative bacteria produce an outer membrane (OM) that envelopes the cell. The OM is an essential organelle and a potent permeability barrier that blocks the entry of many antibiotics into the cell. This barrier limits clinical options for treating Gram-negative infections at a time of escalating resistance to effective therapeutics. The OM is separated from the inner membrane by a periplasmic space. All the components of the OM are delivered across the periplasm to molecular machines that assemble them into the OM bilayer. Lipoproteins (lipid-anchored proteins) are key to OM assembly. Each of the essential OM assembly machines require at least one OM lipoprotein for function. After being secreted from the cytosol, nascent lipoproteins are modified with three acyl chains, anchoring them into the inner membrane. The cell must subsequently traffic these highly hydrophobic lipoproteins through an unfavorable aqueous periplasmic environment towards the OM and anchor them into that membrane. Our goal is to understand the mechanisms of lipoprotein trafficking. The ?Lol pathway? in Escherichia coli has served as the paradigm for how lipoproteins are trafficked by Gram-negative bacteria: the periplasmic protein LolA delivers lipoproteins to the OM where LolB anchors them into the membrane. We discovered recently that while LolA and LolB are important for efficient trafficking, neither protein is essential for lipoprotein trafficking and both proteins can be deleted. In their absence, there is no known mechanism to explain how lipoproteins reach the OM. We aim to uncover the factors behind the LolAB- independent alternate lipoprotein trafficking route. We will pair classical genetics with high-throughput genomics to identify genes that are important for fitness and OM biogenesis when LolAB are deleted. We expect such genes will encode either novel trafficking factors or factors that alleviate stress caused by defects in trafficking. Complementary biochemical studies will aim to directly capture new lipoprotein trafficking factors in action. We also aim to exploit our recent discoveries to offer new insights into LolA and LolB function. Now that we have identified a way to make LolAB conditionally non-essential, we are uniquely placed to thoroughly dissect the molecular mechanisms employed by these proteins. Our studies will broaden the understanding of lipoprotein trafficking in Gram-negative bacteria. Given that lipoproteins are essential for all aspect of OM assembly, our insights will inform the development of novel antibacterial strategies.